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Motivation to obtain preferred foods is enhanced by ghrelin in the ventral tegmental area
Hormones and Behavior 60 (2011) 572–580
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Hormones and Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y h b e h
Motivation to obtain preferred foods is enhanced by ghrelin in the ventral tegmental area S.J. King, A.M. Isaacs, E. O'Farrell, A. Abizaid ⁎ Department of Neuroscience, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6
a r t i c l e
i n f o
Article history: Received 16 May 2011 Revised 11 August 2011 Accepted 12 August 2011 Available online 19 August 2011 Keywords: Ghrelin Reward Ventral tegmental area Food intake Body weight
a b s t r a c t Ghrelin is an orexigenic peptide that acts within the central nervous system to stimulate appetite and food intake via the growth hormone secretagogue receptor (GHS-R). It has been hypothesized that ghrelin modulates food intake in part by stimulating reward pathways in the brain and potentially stimulating the intake of palatable foods. Here we examined the effects of chronic ghrelin administration in the ventral tegmental area (VTA) via osmotic minipumps on 1) ad libitum food intake and bodyweight; 2) macronutrient preference; and 3) motivation to obtain chocolate pellets. In the first study rats receiving ghrelin into the VTA showed a dose-dependent increase in the intake of regular chow, also resulting in increased body weight gain. A second study revealed that intra-VTA delivery of the ghrelin receptor antagonist [Lys-3]-GHRP-6 selectively reduced caloric intake of high-fat chow and reduced body weight gain relative to control and ghrelin treated rats. The third study demonstrated that food restricted rats worked harder for food pellets when infused with ghrelin than when infused with vehicle or ghrelin receptor antagonist treated rats. Finally, rats trained on an FR1 schedule but returned to ad libitum during ghrelin infusion, responded at 86% of baseline levels when they were not hungry, whereas saline infused rats responded at 36% of baseline. Together, these results suggest that ghrelin acts directly on the VTA to increase preference for and motivation to obtain highlypalatable food. © 2011 Elsevier Inc. All rights reserved.
Introduction Ghrelin is a 28 amino-acid peptide that is produced primarily in the X/A-like endocrine cells in the oxyntic glands of the stomach (Date et al., 2000; Kojima et al., 1999), and plays an important role in both the peripheral and central regulation of energy balance. Ghrelin increases food intake and body weight gain when administered systemically, via acute or chronic injections in rats (Tschop et al., 2000; Wren et al., 2000, 2001a). Circulating ghrelin levels are heightened during times of negative energy balance, in both short-term (e.g. fasting) and longterm (e.g. cachexia) conditions (Nagaya et al., 2001; Toshinai et al., 2001). Conversely, plasma ghrelin is decreased during times of abundant energy, such as immediately following a meal, as well as in chronic conditions such as obesity (Tschop et al., 2001). In humans, ghrelin levels show daily rhythms, and peak just prior to voluntary meals (Cummings et al., 2001, 2004). In rodents, peaks are evident just prior to the dark phase of the light cycle, largely coinciding with a typical feeding bout (Drazen et al., 2006). Within the brain, ghrelin binds to the growth hormone secretagogue receptor (GHSR-1a) to increase food intake and adiposity, as well as to decrease the utilization of fat as a source of fuel (Tschop et al., 2000; ⁎ Corresponding author at: Department of Neuroscience, Carleton University, Ottawa, ON, Canada, K1S 5B6. E-mail address: [email protected] (A. Abizaid). 0018-506X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2011.08.006
Zigman et al., 2006). The orexigenic (i.e. appetite stimulating) effects of ghrelin are thought to be mediated mainly through its action on the arcuate nucleus of the hypothalamus (ARC) and brain stem regions such as the nucleus of the solitary tract (Faulconbridge et al., 2003). Both of these regions contribute to homeostatic mechanisms such as meal initiation and a reduction in the utilization of fat for energy demands (Date et al., 2006; Tschop et al., 2000). Within the ARC ghrelin activates orexigenic NPY/AgRP neurons, both of which express GHSR and inhibit central anorexigenic pathways (Nakazato et al., 2001; Tschop et al., 2000; Willesen et al., 1999). While the action of ghrelin on the ARC in the regulation of homeostatic feeding has been well characterized, it does not fully account for the capacity of both rodents and humans to eat beyond their nutritional demands in the presence of appetizing foods. In an effort to address this issue, a rapidly growing body of research suggests that both the direct and indirect action of ghrelin on mesolimbic dopamine (DA) circuitry play a role in both chow intake (Dickson et al., 2010; Naleid et al., 2005) and intake of other highlypalatable options (Disse et al., 2010; Egecioglu et al., 2010). The motivation to obtain highly palatable food sources has also been linked to the action of ghrelin on DA circuitry (Abizaid et al., 2006; Dickson et al., 2010; Perello et al., 2009). In support of this hypothesis, the presence of GHSR message has been reported in the ventral tegmental area (VTA) (Guan et al., 1997; Zigman et al., 2006), a key node in the mesolimbic DA system known
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to be important for regulating responses to reward and reinforcement. Numerous studies have shown that ghrelin binds to the VTA, is colocalized with DA neurons, and a single infusion into the VTA produces a robust feeding response (Abizaid et al., 2006; Naleid et al., 2005). Conversely, blocking VTA ghrelin receptors using a selective GHSR antagonist blunts food intake induced by peripheral ghrelin and rebound feeding in mice fasted overnight (Abizaid et al., 2006). Ghrelin receptor antagonism was also shown to dose-dependently attenuate operant self-administration of a sucrose reward (Landgren et al., 2011; Skibicka et al., 2011, in press), suggesting that the activation of GHSR in the VTA is important for the orexigenic effects of ghrelin, particularly for the hedonic or appetitive responses to a palatable food source. Ghrelin mediates anticipatory feeding responses and also promotes the consumption of foods high in caloric value, particularly high-fat foods (Blum et al., 2009; Clifford et al., 2011; LeSauter et al., 2009; Shimbara et al., 2004). Further, acute ghrelin treatments, both peripheral and intra-VTA, have been shown to increase the effort expended to obtain palatable food rewards using mice trained to perform in operant tasks such as progressive ratios. Alternatively, mice treated with GHSR antagonists or with targeted deletions of GHSRs show reduction in motivation to obtain food rewards (Landgren et al., 2011; Perello et al., 2009; Skibicka et al., 2011, in press). 6hydroxydopamine lesions directed at the VTA also reduced operant responding typically stimulated by central ghrelin administration (Weinberg et al., 2011). It is not known, however, whether the effects of chronic ghrelin administration into the VTA will increase feeding, weight gain, preference for a diet high in fat, and/or an enhanced motivation to eat. It is also unclear how chronic delivery of a ghrelin receptor antagonist into the VTA will affect all of these measures. In the present report we examined these questions and provide additional support for the role of ghrelin acting directly on the VTA to modulate food reward. General method Subjects and housing Male Long-Evans rats (~ 90 days old; n = 94) obtained from Charles River Laboratories (St. Constant, Quebec, Canada) were used for all experiments. Rats were individually housed in transparent Plexiglass cages (48 cm × 26 cm × 20 cm) in a temperature and humidity controlled environment (22 °C and 45–55%, respectively). Rats were left undisturbed for one week to allow for acclimatization to vivarium conditions. All rats were maintained on a standard 12 hour light dark cycle (lights on at 08:00) with ad libitum access to standard laboratory rat chow and water supply, except when otherwise indicated. All experimental procedures complied with the Canadian Council on Animal Care (CCAC) guidelines and were approved by the Local Ethics Committee at Carleton University. Surgical procedures Male rats were anesthetized with isofluorane and oxygen (4:2 for induction; 2:2 for maintenance) for the duration of the procedure. The rats' head and a small dorsal region was shaved and the rat was mounted into a small animal stereotaxic apparatus (Kopf Instruments, Tujunga, CA) positioned atop a heating pad. The scalp was cleaned with both Surgiprep and Priviodine to provide an aseptic canvas; tear gel was also applied to prevent dehydration of the eyes. A midline incision was made using a 10 mm scalpel and the skin and periosteum were retracted to enable clear visualization of bregma. A twenty-six gage stainless steel unilateral guide cannulae (Plastics One, Roanoke, VA) attached via polyethylene catheter to an mini-osmotic pump (Alzet Mini-Osmotic Pump Model 2002; DURECT; flow rate, 0.5 μl/h for 14 days) was implanted into the ventral tegmental area (co-
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ordinates: AP −5.3 mm, ML + 2.0 mm, DV −7.6 mm; Paxinos and Watson, 1998). Mini-pumps were filled with 0.2 ml of either sterile saline (0.9% NaCl), ghrelin (Peptides International; dose range 310 nM/rat/day), or a ghrelin receptor antagonist ([D-Lys3]-GHRP-6) solution (Peptides International; 200 nM/rat/day). Three holes were drilled for implantation of jeweler screws, serving to anchor the cannula to the surface of the skull. The cannula and the screws were affixed to the skull using dental cement. Once the cement was dried completely, the dorsal portion of the skin was separated from the muscle using blunt dissection in order to implant the mini-pump subcutaneously. Following closure of the incision with surgical sutures, Polysporin and Lidocaine were applied to the surgical site to prevent bacterial infection and pain, respectively. Rats were also injected with Metacam (0.1 ml) to provide postoperative analgesia. Upon completion of surgery, rats were placed in clean cages with cob bedding atop a heating pad and maintained in a recovery area until they awoke. At this time, the animals were returned to the vivarium and monitored closely to ensure optimal recovery. Histological analyses On completion of behavioral observations, rats were overdosed with Nembutal and transcardially perfused with 4% paraformaldehyde (PFA). Extracted brains were post-fixed for 24 h in 4% PFA, followed by cryo-protection in a 30% sucrose solution in 0.1 M phosphate buffer solution and storage in 4 °C until sectioning. To verify cannula placements, brains were frozen and sliced into 50 μM coronal sections using a cryostat (LEICA CM1900). Coronal sections were mounted onto gel-coated slides and stained using cresyl violet. Cresyl violet staining Each slide was submerged in the following series of solutions for 2 min each: 100% ethanol, 95% ethanol, and 70% ethanol. Sections were then rinsed in distilled water to remove excess ethanol and subsequently placed in a 1% cresyl violet solution. Sections were then rinsed in distilled water to remove excess cresyl and immersed in 0.8% acetic acid to allow for differentiation (~2–5 min). Sections were then placed in 70%, 95% and 100% ethanol for 2 min each, respectively. Upon completion of staining, slides were immersed in Clearene solution for 15 min prior to cover-slipping with clarion mounting media. Microscopic examination was then used to determine location of infusion sites. Animals with incorrect cannulae placements were not included in the analyses. Experiment 1: the effects of chronic ghrelin on ad libitum food intake and body weight. The main goal of this experiment was to determine the effects of chronic intra-VTA ghrelin on food intake and body weight under standard ad libitum conditions. Twenty-four adult male rats (250–280 g) were single housed and received ad libitum chow and tap water. Following a one week baseline period measuring food intake and body weight, each rat received an indwelling unilateral guide cannula directed at the VTA. Each cannula was connected to an osmotic mini-pump delivering vehicle or one of two doses of ghrelin (3 nM, 10 nM). The rate of delivery was 0.5 μl/h/day for a total of 14 days. Food intake and body weight was monitored every 24 h throughout the infusion period, and for a week after the mini pumps were no longer delivering any drug. Experiment 2: the effects of chronic ghrelin on macronutrient diet preference. Given that ghrelin has been shown to preferentially increase the consumption of a high-fat diet (Shimbara et al., 2004), we hypothesized that ghrelin treated rats would choose high-fat as their primary source of calories, as well as show a substantial increase in body weight, compared to saline controls. Conversely, administration of a ghrelin antagonist was hypothesized to blunt the preference for the high-fat diet in turn causing a reduction in body weight gain.
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Twenty-six adult male rats (250–280 g) were single housed and exposed to three different diets simultaneously present in the home cage: 1) a carbohydrate rich diet, 2) a fat rich diet, and 3) a protein rich diet (Harlan–Teklad Diets; see Table 1 for information on caloric content). After baseline measurement of diet intake and body weight, animals were randomly assigned to 1 of 3 treatment groups and were implanted with 14-day osmotic mini-pumps delivering either 10 nM/ day of ghrelin (Peptides International; n = 9), 200 nM/day of a ghrelin antagonist ([D-Lys3]-GHRP-6; Peptides International; n = 9), or sterile saline (n = 8) into the VTA. Following surgery, daily intake of each macronutrient and body weight gain for the 14 days of treatment and 6 days post-infusion were recorded. Experiment 3: the effects of chronic ghrelin on progressive ratio responding for highly-palatable food pellets. To further examine the influence of ghrelin on the motivation to obtain a highly palatable food source, we measured how much work rats were willing to perform to obtain chocolate-flavored pellets that were isocaloric with our standard lab chow (Bioserv, Product # F0299; 3.68 kcal/g; see Table 2 for detailed content) using a progressive ratio (PR) operant paradigm. In a PR schedule experiment, the response contingency required to obtain a reward is progressively increased after each receipt of reinforcement. This paradigm requires that the animal increases the amount of effort to obtain the same magnitude of reward, thereby increasing the cost/benefit ratio. The efficacy of a given reinforcer (e.g. a food pellet) is defined in terms of the breakpoint (BP), the point in the series at which responding ceases (i.e. the final ratio completed by an animal) and reflects the maximum effort that the animal is willing to exert to obtain a particular reward (Hodos, 1961; Richardson and Roberts, 1996). Twenty-four adult male rats underwent caloric restriction and operant training procedures prior to surgery. To this end, animals were food restricted to 90% of their initial body weight and trained to bar press on a fixed-ratio 1 (FR1) schedule of responding in standard operant conditioning chambers (Colbourn Instruments) equipped with one nose-poke portal flanked by two retractable levers. Every correct press of the active lever resulted in the delivery of one 45 mg chocolate flavored food pellet. Each training session was 30 min in duration and was conducted between 0800 and 1200 h. Once each rat reached a stable level of responding on an FR1 schedule during training, defined as less than 15% variation in number of presses on 2 consecutive days during a 30 minute test session (~ 7 days), stereotaxic surgery was performed. During surgery, rats received minipumps filled with either saline (n = 8), ghrelin (10 nM/day; n = 8), or [D-Lys3]-GHRP-6 (n = 8). Rats were allowed to recover for seven days and tested on a PR schedule of responding on days 8, 10 and 14 following the surgery. The PR schedule required animals to increase the number of responses to obtain one food pellet after every correct response according to the following series: 1, 2, 4, 6, 9, 11, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219 etc. (from Richardson and Roberts, 1996). Table 1 Macronutrient composition of diets used in Experiment 2. Diet
Macronutrient
% by weight
% kcal from
Fat (60%)
Protein Carbohydrates Fat 5.1 Protein Carbohydrates Fat 4 Protein Carbohydrates Fat 3.7
23.5 27.3 34.3
18.4 21.3 60.3
17.7 70 5.2
17.8 70.4 11.8
60 21.1 5.2
64.7 22.7 12.6
Total kcal/g Carbohydrates (70%)
Total kcal/g Protein (60%)
Total kcal/g
Table 2 Macronutrient composition for operant pellets (45 mg). Purified chocolate pellets
Total kcal/g
Nutritional content
% by weight
Protein Carbohydrates Fat Fiber Ash Moisture
18.8 61.5 5.0 4.6 4.4 b5 3.68
We hypothesized that ghrelin treated rats would display an increased motivation to obtain chocolate-flavored pellets, as determined by an increase in breakpoints during treatment. Alternatively, we expected that [D-Lys3]-GHRP-6 treated rats would reduce their level of responding following treatment, suggesting an important role for ghrelin in the motivation to obtain palatable foods. Experiment 4: the effects of chronic intra-VTA ghrelin on operant responding in satiated rats. Here we examined the effects of ghrelin infusions into the VTA on operant responses in animals that were not hungry when tested. Twenty adult male rats (250–280 g) were singlehoused and received ad libitum chow and water during the acclimatization week, during which baseline food intake and body weight were recorded. Rats were subsequently calorically restricted to ~ 50% of their daily food intake until they reached 90% of their body weight. Then, rats received daily 30 minute training sessions until they reached a stable level of responding on a FR1 schedule of reinforcement. Animals were then implanted with intra VTA cannulae attached to mini-pumps filled with either saline (n = 10) or ghrelin (n = 10, 10 nM/day). Following surgical procedures, animals were returned to ad libitum feeding and food intake and body weight were recorded daily. On day 4 post-surgery rats were tested in the operant chambers on an FR 1 schedule to allow comparison to baseline levels of responding, when animals were food restricted. Results Animals in all studies showed minimal discomfort as a result of the surgical procedures and none of the animals had to be discarded due to illness or surgical or post surgical complications. A representative cannulae placement photo and animals that were excluded due to incorrect placements are outlined in Fig. 1. Experiment 1. Ghrelin infused chronically into the VTA increases food intake and body weight gain A one-way between groups ANOVA revealed that by the end of the baseline period, all rats were consuming a similar amount of chow per day (F(2,18) = 1.452, p N 0.05; data not shown) and displayed no significant differences in body weight (F(2,18) = .586, p N 0.05; data not shown). Given these data we organized and analyzed experimental data in the form of change from baseline scores. Following surgery we observed a dose response effect of ghrelin delivered chronically into the VTA on food intake (F(2,18) = 4.337, p b 0.05). Rats receiving the 10 nM dose of ghrelin consumed an average of 6.2 g more chow per day during treatment compared to baseline consumption, whereas saline treated rats consumed only 2.92 g more chow per day (p b 0.05; see Fig. 2a). The group infused with 3 nM ghrelin ate an average of 4.27 g more chow per day, but this change from baseline did not differ significantly from that observed in either high ghrelin (p = .090) or saline groups (p N 0.05; see Fig. 2a). Similarly, chronic intra VTA ghrelin delivery resulted in a significant increase in weight gain from the baseline that was dose dependent (see Fig. 2b). When total weight gain over the infusion period was examined using a one-way ANOVA, rats treated with the
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Fig. 1. Representative photomicrograph of cannula placements in the ventral tegmental area (VTA) and table outlining number of animals discarded from analysis in each experiment based on incorrect placements.
high dose of ghrelin exhibited the greatest total weight gain from baseline (F(2,18) = 3.743, p b 0.05; M = 99.33 g), significantly different from the saline group (p = .021; M = 81.71 g), but not different from the low ghrelin group (p b 0.05; M = 86.75 g; see Fig. 2b).
the preferred high fat diet (F(2,18) = 172.780, p b 0.05), relative to both saline (p b .05) and ghrelin treated rats (p b 0.05 Tukey's HSD, see Fig. 3c). Neither ghrelin nor [D-Lys3]-GHRP-6 infusions affected the intake of the protein or carbohydrate rich diet (p N 0.05; See Fig. 3c).
Experiment 2. GHSR blockade in the VTA decreases the intake of food with a high caloric content coming from fat
Experiment 3. Ghrelin infusions into the VTA enhance, while GHSR blockade prevents increases in food motivation as measured by progressive ratio responding under restricted feeding conditions
As in the previous study, there were no differences in food intake or body weight between the groups during the baseline period (p N 0.05; data not shown). It should be noted that all animals showed a clear preference for the diet with high caloric content coming from fat (Fig. 3a). In addition, access to a choice in diets resulted in high caloric intake, and a rapid gain in weight in all animals (Raynor and Epstein, 2001), in comparison with similar baseline measures from animals in the first experiment (See Table 3). Interestingly, while intra-VTA ghrelin did not further increase neither caloric intake nor body weight gain in this experiment, the blockade of ghrelin receptors in the VTA with the ghrelin antagonist [D-Lys3]-GHRP-6 selectively attenuated their body weight gain (F(2,18) = 9.32, p b 0.05 Tukey's HSD; see Fig. 3b) and their intake of
A one way ANOVA showed that rats in each group had similar body weights before any experimental manipulations were conducted (F(2,19) = .812, p N 0.05; data not shown). Similarly, there were no differences between rats in the time it took to acquire a stable rate of operant responding during the training period (p N 0.05). Following surgery, animals were tested under the progressive ratio schedule of responding at three different time points during the infusion period: 1) On day 8 of the infusion period (day 1 of food restriction); 2) day 11 of the infusion period (day 4 food restriction); and 3) day 14 of the infusion period (day 7 of food restriction). All animals were tested under restricted feeding conditions that began on the evening of day 7 postinfusion. As shown by repeated measures ANOVA (time interval x group), by day 14 there was an overall treatment effect where ghrelin treated rats responded more than saline or antagonist treated rats (main effect for treatment, F(1,19) = 3.741, p b .05). As expected, efforts to obtain pellets were increased over time following the food restriction regimen (main effect for time interval, F(1,19) = 22.798, p b 0.05). a significant interaction effect emerged, demonstrating that rats infused with the ghrelin receptor antagonist into the VTA, failed to increase their efforts to obtain pellets over time in spite of being food restricted, in comparison to ghrelin treated animals (F(2,19) = 6.599, p b 0.05, followed by post hoc Tukey's HSD: p b 0.05; see Fig. 4). The data were also analyzed in terms of break points, defined as the last completed (reinforced) ratio before the animal stopped responding. As shown in Fig. 5, a repeated measures ANOVA revealed no differences between the groups on day 1 or day 4 following the onset of food restriction (p N 0.05; data not shown). However, during the PR session on day 7, differences were evident between groups (F(2,19) = 7.398, p b .05), with the ghrelin infused rats showing an increase in effort spent to obtain chocolate-flavored pellets relative to the [D-Lys3]-GHRP-6 group (p b 0.05). Ghrelin treated rats pressed the reinforcing lever 167 times on average to receive one chocolate pellet, whereas [D-Lys3]-GHRP-6 treated rats pressed the lever only 42.5 times on average to receive a similar reward. Experiment 4. Ghrelin infusions into the VTA produce “hungry-like” rates of reinforcement in satiated animals
Fig. 2. Mean (+/−SEM) change from baseline on a) daily food intake and b) body weight gain per day in animals infused with either saline (n = 7), 3 nM ghrelin (n = 8), or 10 nM ghrelin (n = 6) during the infusion period (n = 6). * p b .05.
Animals trained to bar press for food under restricted feeding conditions were implanted with cannulae delivering saline or ghrelin
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Fig. 3. Panel a depicts the Mean (+/−SEM) daily kilocalorie consumption as a function of macronutrient diet during the baseline period. All animals exhibited a clear preference for the diet with high caloric content coming from fat over the other diets. Panel b depicts weight gain during the infusion period, clearly showing an attenuation in weight gain in animals infused with [D-Lys3]-GHRP-6. * p b .05. Panel c shows the mean (+/−SEM) daily caloric intake of the high fat, high protein, high carbohydrate diet, and the total caloric intake during the infusion period. ([D-Lys3]-GHRP-6: n = 8; saline: n = 6; ghrelin: n = 8). As seen in this panel, GHSR antagonism with [D-Lys3]-GHRP-6 attenuated high fat intake throughout the infusion period relative to control and ghrelin treated rats. No differences were found between groups on measures of high-protein or high-carbohydrate diets during treatment (p b 0.05).
(10 nM) into the VTA. By the end of infusion period, ghrelin treated rats had consumed more chow during the treatment period compared to saline treated rats (t(1,15) = −3.170, p b .05; Fig. 5a), consistent with greater total body weight gain over the two week period (t(1,15) = −2.195, p b .05; Fig. 6) (see Fig. 6b). When tested in the operant boxes, ghrelin treated rats with full access to food at all times after surgery showed a rate of responding that Table 3 Comparison of daily caloric intake and bodyweight during baseline.
Experiment 1 (standard chow) Experiment 2 (3 diets)
Daily chow intake (kcal)
Weight gain
98.45 +/− 1.789 135.89 +/− 3.65
70.38 +/− 1.81 41.63 +/− 2.72
was nearly equal (about 86%) to that of their baseline rates during which they were food restricted. In contrast, saline treated rats responded at a much lower rate (about 34%) of their baseline “hungry” rate on an FR1 schedule. The difference between ghrelin and vehicle treated rats was statistically significant (t(1,15) = −2.558, p b .05; see Fig. 6c). Discussion In the present series of studies we examined the VTA as a target for ghrelin to enhance the intake of and motivation to obtain palatable food. Using mini-osmotic pumps we chronically infused ghrelin into the VTA of rats, and this treatment resulted in a dose dependant increase in food intake and body weight compared to vehicle infused rats. Furthermore, chronic infusions of a ghrelin antagonist into the
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Fig. 4. Mean (+/−SEM) cumulative bar presses throughout each of the progressive ratio testing sessions during the infusion period. ([D-Lys3]-GHRP-6: n = 7; saline: n = 8; ghrelin: n = 7). By the last day of testing, ghrelin treated animals were pressing the active lever more frequently than both the antagonist and saline treated rats. Antagonist treated rats failed to show an increase in responding across the infusion period compared to ghrelin treated rats, despite both groups being food restricted.
VTA selectively decreased the intake of a preferred high-fat diet, and attenuated the extreme body weight gain that is seen in control animals that had access to a variety of diets. Chronic VTA infusions of the antagonist also decreased motivation to work for chocolate flavored (but not calorie rich) pellets in hungry animals, whereas ghrelin further enhanced motivation to work for these pellets in hungry animals. Finally, animals receiving ghrelin infusions into the VTA responded like hungry animals when tested on an FR1 schedule under ad lib conditions.
Fig. 5. Amount of work rats were willing to perform as measured by Mean (+/−SEM) breakpoints as observed in each progressive ratio testing session. During the final progressive ratio test ghrelin treated rats expended greater effort to obtain chocolate pellets relative to the antagonist treated rats. * p b .05.
Previous studies have shown that chronic ghrelin infusions, both peripheral and central (intracerebroventricular), result in increased food intake, body weight and adiposity in rodents (Perez-Tilve et al., 2011; Tschop et al., 2000). While these effects have been generally attributed to the actions of ghrelin on receptors within the hypothalamic arcuate nucleus, our current data suggest that chronic VTA stimulation with ghrelin can produce similar results. In fact, the body weight gain and food intake seen in our study following chronic VTA infusions of ghrelin, are very similar to those reported by Tschop et al. (2000) in rats chronically infused with ghrelin into the ventricles. These data further support a number of recent studies demonstrating that acute ghrelin infusions directed as the VTA results in an increased motivation for palatable food in mice as measured by CPP and operant paradigms (Egecioglu et al., 2010; Landgren et al., 2011; Skibicka et al., 2011, in press). While these data do not imply that the VTA is uniquely responsible for the obesogenic effects of ghrelin, they do indicate that the VTA could play an important role in the regulation of food intake and body weight. These data also confirm that ghrelin stimulates appetite by acting directly on the VTA (Abizaid et al., 2006; Naleid et al., 2005). Furthermore, these data suggest that these effects can persist in spite of chronic stimulation of GHSRs. This was clearly evident in rats given the highest dose of ghrelin, who were eating about 30% (an average of 9 g of food) more on the last day of ghrelin infusion than their baseline average intake. Control animals also increased their intake of food (something that is natural in animals that are growing), but this increase was less substantial at 12% (4.5 g on average). Thus, even in the absence of caloric restriction, chronically elevated ghrelin can serve to induce a sustained feeding response when administered into the VTA.
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Fig. 6. Mean (+/−SEM) a) body weight gain; b) cumulative chow consumption, and c) percent of baseline responding following surgery on an FR1 schedule of reinforcement during the infusion period (Saline: n = 7; Ghrelin: n = 10). Ghrelin treated rats gained more weight, ate more, and bar-pressed for food more under ad lib conditions than saline infused animals. * p b .05.
Our study supports the notion that ghrelin receptors in the VTA mediate the consumption of preferred foods, whether the preference is for calorie rich high fat diet or an isocaloric chocolate flavored food pellet. We found, as others have previously (see Raynor and Epstein, 2001 for review), that our rats consumed a large amount of calories (approximately 70% more) and gained more weight when given access to a variety of foods compared to rats given regular chow. The rats in our study showed a strong preference for the food with the highest percentage of calories coming from fat. While ghrelin infusions into the VTA were not effective in further increasing neither total caloric intake, nor the intake of the high fat selectively, infusions of the ghrelin antagonist selectively reduced the intake of this high fat diet without altering the intake of the other two diets. Similarly, while intra VTA ghrelin did not further affect weight gain, the antagonist attenuated weight gain during the infusion period. It should be noted, however, that the antagonist utilized in this series of studies has been shown to be less specific to the GHSR than some alternatives (Depoortere et al., 2006). Nevertheless, our results are consistent
with those of others using different antagonists to reduce behavioral responses to obtain food and other reinforcers (Egecioglu et al., 2010; Landgren et al., 2011; Skibicka et al., 2011, in press). Previous human and animal studies have shown that ghrelin not only increases food intake but also that, when given a choice, ghrelin treatment increases the intake of preferred foods via central mechanisms. For example, ICV ghrelin infusions increased the intake of a highfat diet that was presented to rats simultaneously with a highcarbohydrate diet (Shimbara et al., 2004). In rats and mice, ghrelin injections increase the intake of sweet tasting solutions regardless of caloric content, an effect not seen in ghrelin insensitive mice (Disse et al., 2010). In contrast, ghrelin receptor antagonists decrease the intake of palatable liquid diets in rats (e.g. Ensure™), while acute intra VTA infusions increase the intake of these same diets (Egecioglu et al., 2010). Human studies have shown that hunger scores and food intake increase after ghrelin injections (Wren et al., 2001b), and that ghrelin can selectively increase imagery of preferred foods regardless of the type of food (Schmid et al., 2005). Moreover, ghrelin stimulates the mesolimbic reward system of human subjects that are shown images of food (Malik et al., 2008). It is likely that ghrelin acts on the VTA to increase the incentive value of food, particularly preferred foods if these are present. Further, our data suggest that the VTA may be a potential pharmaceutical target for ghrelin related drugs to reduce overconsumption of palatable foods, or increase food intake in clinical conditions where this is required. Given that systemically and centrally administered ghrelin acts on the VTA to stimulate DA cells and increases extracellular concentration of DA in the NAc (Abizaid et al., 2006; Jerlhag et al., 2007; Quarta et al., 2009), we hypothesized that ghrelin administration into the VTA would increase instrumental responses for a reward. Our results confirmed this. In Experiment 3 we show that vehicle treated animals generally increased the number of responses they were willing to perform as the duration of food restriction increased. Ghrelin infused rats showed similar responses as vehicle treated rats after one and four days of food restriction, but were more willing to continue responding in the face of increased demands seven days after the food restriction began. In contrast, animals chronically infused with the ghrelin antagonist never increased their willingness to respond to the increased demands posed by the progressive ratio schedule. Interestingly, even after seven days of food restriction, antagonist treated rats quit responding sooner than vehicle treated rats that, in turn, had a shorter latency to quit responding than ghrelin treated rats. While we did not observe a ghrelin effect on PR responses when rats were not food restricted, we did observe that ghrelin infused into the VTA made satiated rats respond at the same rate or higher than they did when they were hungry under an F1 schedule. In contrast, vehicle treated rats responded only at about 60% of their hungry rates, indicating that ghrelin may act on the VTA to produce some behavioral responses in satiated animals that are akin to those seen in hungry animals. The role of the VTA in the modulation of feeding is complex, and appears to be closely linked to the motivational aspects of feeding, in addition to influencing the hunger/satiety pathway that is generally governed by the hypothalamus. The relative contribution of the hypothalamus and the VTA in the central mechanisms that govern feeding behavior and motivation remains to be fully determined. Nevertheless, it is possible that ghrelin receptors in the VTA enhance information that is relayed to the VTA via a number of ascending and descending pathways that have been implicated in arousal, sensory information, and reward. For instance, ghrelin receptors in the VTA may facilitate the activation of DA neurons by orexin, a lateral hypothalamic peptide that generates arousal and has been linked to reward seeking behaviors (Aston-Jones et al., 2010; Boutrel et al., 2010; Thompson and Borgland, 2010). Similarly, ghrelin receptors in the VTA may lower the threshold of activation mediated by glutamatergic and/or cholinergic inputs to the VTA and influence the intake of rewarding food (Abizaid et al., 2006; Jerlhag et al., 2006).
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Moreover, recent reports point to the importance of the interactions between cholinergic pathways and ghrelin signaling pathways to influence the VTA (Dickson et al., 2010; Disse et al., 2011). The experimental approach used in the present study allowed us to deliver ghrelin or ghrelin antagonists directly onto the VTA of rats at a constant rate for a period of 14 days. Postmortem histological examination determined that most of the animals in each study had cannulae that were accurate in their placement. Indeed, very few animals per experiment had a cannula that was in areas surrounding the VTA, and these animals were discarded from further analyses. We cannot discard the possibility that the observed effects of the different drugs infused with the minipumps are due at least in part to the actions of these drugs spilling into nearby areas. Previous studies, however, have shown that acute infusions of ghrelin at a rate of 0.5 μl infused over a period of two minutes into the rat VTA do not produce spillage outside of the VTA (Abizaid et al., 2006). Furthermore, in these same acute feeding experiments, ghrelin infusions into areas surrounding the VTA failed to produce any significant effects on feeding (Abizaid et al., 2006). Thus, given these data we attribute our results to the effects of ghrelin or its antagonists to their actions on GHSR within the VTA. As chronic overeating and the resultant obesity that often accompanies it has become an increasing threat to the health of individuals worldwide, it is important to determine what can be done to reduce it. As we have shown, antagonism of ghrelin not only reduced body weight gain and preference for a high-fat diet when no effort was required, it also blunted the effect of ghrelin on the effort expended to obtain flavored food pellets. Emerging evidence, including the current study, suggests that the ghrelin system may provide a potential therapeutic target for the treatment of obesity or wasting disorders. Acknowledgments This project was funded by a Natural Science and Engineering Research Council of Canada (NSERC) Discovery grant (AA), an Ontario Graduate Scholarship (SJK), and NSERC summer undergraduate scholarships (EO and AI). References Abizaid, A., Liu, Z., Andrews, Z.B., Shanabrough, M., Borok, E., Elsworth, J.D., Roth, R.H., Sleeman, M.W., Picciotto, M.R., Tschop, M., Gao, X., Horvath, T., 2006. Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. J. Clin. Invest. 116 (12), 3229–3239. Aston-Jones, G., Smith, R.J., Sartor, G.C., Moorman, D.E., Massi, L., Tahsili-Fahadan, P., Richardson, K.A., 2010. Lateral hypothalamic orexin/hypocretin neurons: a role in reward-seeking and addiction. Brain Res. 1314, 74–90. Blum, I.D., Patterson, Z., Khazall, R., Lamont, E.W., Sleeman, M.W., Horvath, T.L., Abizaid, A., 2009. Reduced anticipatory locomotor responses to scheduled meals in ghrelin receptor deficient mice. Neuroscience 164 (2), 351–359. Boutrel, B., Cannella, N., de Lecea, L., 2010. The role of hypocretin in driving arousal and goal directed behaviors. Brain Res. 1314, 103–111. Clifford, S., Zeckler, R.A., Buckman, S., Thompson, J., Wellman, P.J., Smith, R.G., 2011. Impact of food restriction and cocaine on locomotion in ghrelin- and ghrelinreceptor knockout mice. Addict. Biol. 16 (3), 386–392 (Jul). Cummings, D.E., Purnell, J.Q., Frayo, R.S., Schmidova, K., Wisse, B.E., Weigle, D.S., 2001. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 50, 1714–1719. Cummings, D.E., Frayo, R.S., Marmonier, C., Aubert, R., Chapelot, D., 2004. Plasma ghrelin levels and hunger scores in humans initiating meals voluntarily without time- and food-related cues. Am. J. Physiol. Endocrinol. Metab. 287, 297–304. Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., Suganuma, T., Matsukura, S., Kangawa, K., Nakazato, M., 2000. Ghrelin, a novel growth hormone releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141 (11), 4255–4261. Date, Y., Shimbara, T., Koda, S., Toshinai, K., Ida, T., Murakami, N., Miyazato, M., Kokame, K., Ishizuka, Y., Ishida, Y., Kageyama, H., Shioda, S., Kangawa, K., Nakazoto, M., 2006. Peripheral ghrelin transmits orexigenic signals through the noradrenergic pathway from the hindbrain to the hypothalamus. Cell Metab. 4, 323–331. Depoortere, I., Thijs, T., Peeters, T., 2006. The contractile effect of the ghrelin receptor antagonist, D-Lys-3 [GHRP-6], in rat fundic strips is mediated through 5-HT receptors. Eur. J. Pharmacol. 537, 160–165. Dickson, S.L., Hrabovsky, E., Hansson, C., Jerlhag, E., Alvarez-Crepo, M., Skibicka, K.P., Molnar, C.S., LIposits, Z., Engel, J., Egecioglu, E., 2010. Blockade of central nicotine
579
acetylcholine receptor signaling attenuates ghrelin-induced food intake in rodents. Neuroscience 171, 1180–1186. Disse, E., Bussier, A.L., Veyrat-Durebex, C., Deblon, N., Pfluger, P.T., Tschop, M.H., Laville, M., Rohner-Jeanrenaud, F., 2010. Peripheral ghrelin enhances sweet taste food consumption and preference, regardless of its caloric content. Physiol. Behav. 101 (2), 277–281. Disse, E., Bussier, A.L., Deblon, N., Pfluger, P.T., Tschop, M.H., Laville, M., RohnerJeanrenaud, F., 2011. Systemic ghrelin and reward: effect of cholinergic blockade. Physiol. Behav. 102, 481–484. Drazen, D.L., Vahl, T.P., D'Alessio, D.A., Seeley, R.J., Woods, S.C., 2006. Effects of a fixed meal pattern on ghrelin secretion: evidence for a learned response independent of nutrient status. Endocrinology 147 (1), 23–30. Egecioglu, E., Jerlhag, E., Salome, N., Skibicka, K.P., Haage, D., Bohlooly-Y, M., Andersson, D., Bjursell, M., Perrissoud, D., Engel, J., Dickson, S.L., 2010. Ghrelin increases intake of rewarding food in rodents. Addict. Biol. 15 (3), 304–311. Faulconbridge, L.F., Cummings, D.E., Kaplan, J.M., Grill, H.J., 2003. Hyperphagic effects of brainstem ghrelin administration. Diabetes 52, 2260–2265. Guan, X.M., Yu, H., Palyha, O.C., McKee, K.K., Feighner, S.D., Sirinathsinghji, D.J., Smith, R. G., Van der Ploeg, L.H.T., Howard, A.D., 1997. Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Mol. Brain Res. 48, 23–29. Hodos, W., 1961. Progressive Ratio as a measure of reward strength. Science 134, 943–944. Jerlhag, E., Egecioglu, E., Landgren, S., Salome, N., Heilig, M., Moechars, D., Datta, R., Perrissoud, D., Dickson, S.L., Engel, J.A., 2006. Ghrelin stimulates locomotor activity and accumbal dopamine-overflow via central cholinergic systems in mice: implications for its involvement in brain reward. Addict. Biol. 11 (1), 45–54 (Mar). Jerlhag, E., Egecioglu, E., Dickson, S.L., Douhan, A., Svensson, L., Engel, J.A., 2007. Ghrelin administration into tegmental areas stimulates locomotor activity and increases extracellular concentration of dopamine in the nucleus accumbens. Addict. Biol. 12, 6–16. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone releasing acylated peptide from stomach. Nature 402, 656–660. Landgren, S., Simms, J.A., Thelle, D.S., Strandhagen, E., Bartlett, S.E., Engel, J.A., Jerlhag, E., 2011. The ghrelin signalling system is involved in the consumption of sweets. PLoS One 6 (3), e18170. LeSauter, J., Hoque, N., Weintraub, N., Pffaf, D.W., Silver, R., 2009. Stomach ghrelinsecreting cells as food-entrainable circadian clocks. PNAS 106 (32), 13582–13587. Malik, S., McGlone, F., Bedrossian, D., Dagher, A., 2008. Ghrelin modulates brain activity in areas that control appetitive behavior. Cell Metab. 7 (5), 400–409. Nagaya, N., Uematsu, M., Kojima, M., Date, Y., Nakazato, M., Okumura, H., Hosoda, H., Shimizu, W., Yamagishi, M., Oya, H., Yutani, C., Kangawa, K., 2001. Elevating circulating levels of ghrelin in cachexia associated with chronic heart failure: relationships between ghrelin and anabolic/catabolic factors. Circulation 104, 2034–2038. Nakazato, M., Murakami, N., Date, Y., Kojima, M., Matsuo, H., Kangawa, K., Matsukura, S., 2001. A role for ghrelin in the central regulation of feeding. Nature 409, 194–198. Naleid, A.M., Grace, M.K., Cummings, D.E., Levine, A.S., 2005. Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens. Peptides 26, 2274–2279. Paxinos, G., Watson, C., 1998. The rat brain in stereotaxic coordinates, 4th edition. Academic Press, San Diego, CA. Perello, M., Sakata, I., Birnbaum, S., Chuang, J.C., Osbourne-Lawrence, S., Rovinsky, S.A., Woloszyn, J., Yanagisawa, M., Lutter, M., Zigman, J., 2009. Ghrelin increases the rewarding value of high-fat diet in an orexin dependent fashion. Biol. Psychiatry 67 (9), 880–886. Perez-Tilve, D., Heppner, K., Kirchner, F., Lockie, S.H., Woods, S.C., Smiley, D.L., Tschop, M., Pfluger, P., 2011. Ghrelin induced adiposity is independent of orexigenic effects. FASEB J. 25 (8), 2814–2822 (Aug). Quarta, D., DiFrancesco, C., Melotto, S., Mangiarini, L., Heidbreder, C., Hedou, G., 2009. Systemic administration of ghrelin increases extracellular dopamine in the shell but not the core subdivision of the nucleus accumbens. Neurochem. Int. 54 (2), 89–94. Raynor, H.A., Epstein, L.H., 2001. Dietary variety, energy regulation and obesity. Psychol. Bull. 127 (3), 325–341. Richardson, N.R., Roberts, D.C.S., 1996. Progressive ratio schedules in drug selfadministration studies in rats: a method to evaluate reinforcing efficacy. J. Neurosci. Methods 66, 1–11. Schmid, D.A., Held, K., Ising, M., Uhr, M., Weickel, J.C., Steiger, A., 2005. Ghrelin stimulates appetite, imagination of food, GH, ACTH, and cortisol, but does not affect leptin in normal controls. Neuropsychopharmacology 30, 1187–1192. Shimbara, T., Mondal, M.S., Kawagoe, T., Toshinai, K., Koda, S., Yamaguchi, H., Date, Y., Nakazato, M., 2004. Central administration of ghrelin preferentially increases fat ingestion. Neurosci. Lett. 369, 75–79. Skibicka, K.P., Hansson, C., Alvarez-Crespo, M., Friberg, P.A., Dickson, S.L., 2011. Ghrelin directly targets the ventral tegmental area to increase food motivation. Neuroscience 180, 129–137 (Apr 28). Skibicka, K.P., Hansson, C., Egecioglu, E., Dickson, S.L., in press. Role of ghrelin in food reward: impact of ghrelin on sucrose self-administration and mesolimbic dopamine and acetycholine receptor gene expression. Addict. Biol. doi:10.1111/j. 1369-1600.2010.00294.x. Thompson, J.L., Borgland, S.L., 2010. A role for orexin/hypocretin in motivation. Behav. Brain Res. 217, 446–453. Toshinai, K., Mondal, M.S., Nakazato, M., Date, Y., Murakami, N., Kojima, M., Kangawa, K., Matsukura, S., 2001. Upregulation of ghrelin expression in the stomach upon
580
S.J. King et al. / Hormones and Behavior 60 (2011) 572–580
fasting, insulin induced hypoglycemia and leptin administration. Biochem. Biophys. Res. Commun. 281 (5), 1220–1225. Tschop, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908–913. Tschop, M., Wawarta, R., Reipel, R.L., Freidrich, S., Bindlingmaier, M., Landgraf, R., Folwaczny, C., 2001. Postprandial decrease of circulating ghrelin human ghrelin levels. J. Endocrinol. Invest. 24 (6), 19–21. Weinberg, Z.Y., Nicholson, M.L., Currie, P.J., 2011. 6-hydroxydopamine lesions of the ventral tegmental area suppress ghrelin's ability to elicit food reinforced behavior. Neurosci. Lett. 499, 70–73. Willesen, M.G., Kristensen, P., Romer, J., 1999. Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Neuroendocrinology 70 (5), 306–316.
Wren, A.M., Small, C.J., Ward, H.L., Murphy, K.G., Dakin, C.L., Taheri, S., Kennedy, A.R., Roberts, G.H., Morgan, D.G., Ghatei, M.A., Bloom, S.R., 2000. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Reg. Pept. 140, 148–152. Wren, A.M., Seal, L.J., Cohen, M.A., Brynes, A.E., Frost, G.S., Murphy, K.G., Dhillo, W.S., Ghatei, M.A., Bloom, S.R., 2001a. Ghrelin enhances appetite and increases food intake in humans. J. Clin. Endocrinol. Metab. 86 (12), 5992–5995. Wren, A.M., Small, C.J., Abbott, C.R., Dhillo, W.S., Seal, L.J., Cohen, M.A., Batterham, R.L., Taheri, S., Stanley, S.A., Ghatei, M.A., Bloom, S.R., 2001b. Ghrelin causes hyperphagia and obesity in rats. Diabetes 50, 2540–2547. Zigman, J.M., Jones, J.E., Lee, C.E., Saper, C.B., Elmquist, J.K., 2006. Expression of the ghrelin receptor mRNA in the rat and the mouse brain. J. Comp. Neurol. 494, 528–548.
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Hormones and Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y h b e h
Motivation to obtain preferred foods is enhanced by ghrelin in the ventral tegmental area S.J. King, A.M. Isaacs, E. O'Farrell, A. Abizaid ⁎ Department of Neuroscience, Carleton University, 1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6
a r t i c l e
i n f o
Article history: Received 16 May 2011 Revised 11 August 2011 Accepted 12 August 2011 Available online 19 August 2011 Keywords: Ghrelin Reward Ventral tegmental area Food intake Body weight
a b s t r a c t Ghrelin is an orexigenic peptide that acts within the central nervous system to stimulate appetite and food intake via the growth hormone secretagogue receptor (GHS-R). It has been hypothesized that ghrelin modulates food intake in part by stimulating reward pathways in the brain and potentially stimulating the intake of palatable foods. Here we examined the effects of chronic ghrelin administration in the ventral tegmental area (VTA) via osmotic minipumps on 1) ad libitum food intake and bodyweight; 2) macronutrient preference; and 3) motivation to obtain chocolate pellets. In the first study rats receiving ghrelin into the VTA showed a dose-dependent increase in the intake of regular chow, also resulting in increased body weight gain. A second study revealed that intra-VTA delivery of the ghrelin receptor antagonist [Lys-3]-GHRP-6 selectively reduced caloric intake of high-fat chow and reduced body weight gain relative to control and ghrelin treated rats. The third study demonstrated that food restricted rats worked harder for food pellets when infused with ghrelin than when infused with vehicle or ghrelin receptor antagonist treated rats. Finally, rats trained on an FR1 schedule but returned to ad libitum during ghrelin infusion, responded at 86% of baseline levels when they were not hungry, whereas saline infused rats responded at 36% of baseline. Together, these results suggest that ghrelin acts directly on the VTA to increase preference for and motivation to obtain highlypalatable food. © 2011 Elsevier Inc. All rights reserved.
Introduction Ghrelin is a 28 amino-acid peptide that is produced primarily in the X/A-like endocrine cells in the oxyntic glands of the stomach (Date et al., 2000; Kojima et al., 1999), and plays an important role in both the peripheral and central regulation of energy balance. Ghrelin increases food intake and body weight gain when administered systemically, via acute or chronic injections in rats (Tschop et al., 2000; Wren et al., 2000, 2001a). Circulating ghrelin levels are heightened during times of negative energy balance, in both short-term (e.g. fasting) and longterm (e.g. cachexia) conditions (Nagaya et al., 2001; Toshinai et al., 2001). Conversely, plasma ghrelin is decreased during times of abundant energy, such as immediately following a meal, as well as in chronic conditions such as obesity (Tschop et al., 2001). In humans, ghrelin levels show daily rhythms, and peak just prior to voluntary meals (Cummings et al., 2001, 2004). In rodents, peaks are evident just prior to the dark phase of the light cycle, largely coinciding with a typical feeding bout (Drazen et al., 2006). Within the brain, ghrelin binds to the growth hormone secretagogue receptor (GHSR-1a) to increase food intake and adiposity, as well as to decrease the utilization of fat as a source of fuel (Tschop et al., 2000; ⁎ Corresponding author at: Department of Neuroscience, Carleton University, Ottawa, ON, Canada, K1S 5B6. E-mail address: [email protected] (A. Abizaid). 0018-506X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2011.08.006
Zigman et al., 2006). The orexigenic (i.e. appetite stimulating) effects of ghrelin are thought to be mediated mainly through its action on the arcuate nucleus of the hypothalamus (ARC) and brain stem regions such as the nucleus of the solitary tract (Faulconbridge et al., 2003). Both of these regions contribute to homeostatic mechanisms such as meal initiation and a reduction in the utilization of fat for energy demands (Date et al., 2006; Tschop et al., 2000). Within the ARC ghrelin activates orexigenic NPY/AgRP neurons, both of which express GHSR and inhibit central anorexigenic pathways (Nakazato et al., 2001; Tschop et al., 2000; Willesen et al., 1999). While the action of ghrelin on the ARC in the regulation of homeostatic feeding has been well characterized, it does not fully account for the capacity of both rodents and humans to eat beyond their nutritional demands in the presence of appetizing foods. In an effort to address this issue, a rapidly growing body of research suggests that both the direct and indirect action of ghrelin on mesolimbic dopamine (DA) circuitry play a role in both chow intake (Dickson et al., 2010; Naleid et al., 2005) and intake of other highlypalatable options (Disse et al., 2010; Egecioglu et al., 2010). The motivation to obtain highly palatable food sources has also been linked to the action of ghrelin on DA circuitry (Abizaid et al., 2006; Dickson et al., 2010; Perello et al., 2009). In support of this hypothesis, the presence of GHSR message has been reported in the ventral tegmental area (VTA) (Guan et al., 1997; Zigman et al., 2006), a key node in the mesolimbic DA system known
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to be important for regulating responses to reward and reinforcement. Numerous studies have shown that ghrelin binds to the VTA, is colocalized with DA neurons, and a single infusion into the VTA produces a robust feeding response (Abizaid et al., 2006; Naleid et al., 2005). Conversely, blocking VTA ghrelin receptors using a selective GHSR antagonist blunts food intake induced by peripheral ghrelin and rebound feeding in mice fasted overnight (Abizaid et al., 2006). Ghrelin receptor antagonism was also shown to dose-dependently attenuate operant self-administration of a sucrose reward (Landgren et al., 2011; Skibicka et al., 2011, in press), suggesting that the activation of GHSR in the VTA is important for the orexigenic effects of ghrelin, particularly for the hedonic or appetitive responses to a palatable food source. Ghrelin mediates anticipatory feeding responses and also promotes the consumption of foods high in caloric value, particularly high-fat foods (Blum et al., 2009; Clifford et al., 2011; LeSauter et al., 2009; Shimbara et al., 2004). Further, acute ghrelin treatments, both peripheral and intra-VTA, have been shown to increase the effort expended to obtain palatable food rewards using mice trained to perform in operant tasks such as progressive ratios. Alternatively, mice treated with GHSR antagonists or with targeted deletions of GHSRs show reduction in motivation to obtain food rewards (Landgren et al., 2011; Perello et al., 2009; Skibicka et al., 2011, in press). 6hydroxydopamine lesions directed at the VTA also reduced operant responding typically stimulated by central ghrelin administration (Weinberg et al., 2011). It is not known, however, whether the effects of chronic ghrelin administration into the VTA will increase feeding, weight gain, preference for a diet high in fat, and/or an enhanced motivation to eat. It is also unclear how chronic delivery of a ghrelin receptor antagonist into the VTA will affect all of these measures. In the present report we examined these questions and provide additional support for the role of ghrelin acting directly on the VTA to modulate food reward. General method Subjects and housing Male Long-Evans rats (~ 90 days old; n = 94) obtained from Charles River Laboratories (St. Constant, Quebec, Canada) were used for all experiments. Rats were individually housed in transparent Plexiglass cages (48 cm × 26 cm × 20 cm) in a temperature and humidity controlled environment (22 °C and 45–55%, respectively). Rats were left undisturbed for one week to allow for acclimatization to vivarium conditions. All rats were maintained on a standard 12 hour light dark cycle (lights on at 08:00) with ad libitum access to standard laboratory rat chow and water supply, except when otherwise indicated. All experimental procedures complied with the Canadian Council on Animal Care (CCAC) guidelines and were approved by the Local Ethics Committee at Carleton University. Surgical procedures Male rats were anesthetized with isofluorane and oxygen (4:2 for induction; 2:2 for maintenance) for the duration of the procedure. The rats' head and a small dorsal region was shaved and the rat was mounted into a small animal stereotaxic apparatus (Kopf Instruments, Tujunga, CA) positioned atop a heating pad. The scalp was cleaned with both Surgiprep and Priviodine to provide an aseptic canvas; tear gel was also applied to prevent dehydration of the eyes. A midline incision was made using a 10 mm scalpel and the skin and periosteum were retracted to enable clear visualization of bregma. A twenty-six gage stainless steel unilateral guide cannulae (Plastics One, Roanoke, VA) attached via polyethylene catheter to an mini-osmotic pump (Alzet Mini-Osmotic Pump Model 2002; DURECT; flow rate, 0.5 μl/h for 14 days) was implanted into the ventral tegmental area (co-
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ordinates: AP −5.3 mm, ML + 2.0 mm, DV −7.6 mm; Paxinos and Watson, 1998). Mini-pumps were filled with 0.2 ml of either sterile saline (0.9% NaCl), ghrelin (Peptides International; dose range 310 nM/rat/day), or a ghrelin receptor antagonist ([D-Lys3]-GHRP-6) solution (Peptides International; 200 nM/rat/day). Three holes were drilled for implantation of jeweler screws, serving to anchor the cannula to the surface of the skull. The cannula and the screws were affixed to the skull using dental cement. Once the cement was dried completely, the dorsal portion of the skin was separated from the muscle using blunt dissection in order to implant the mini-pump subcutaneously. Following closure of the incision with surgical sutures, Polysporin and Lidocaine were applied to the surgical site to prevent bacterial infection and pain, respectively. Rats were also injected with Metacam (0.1 ml) to provide postoperative analgesia. Upon completion of surgery, rats were placed in clean cages with cob bedding atop a heating pad and maintained in a recovery area until they awoke. At this time, the animals were returned to the vivarium and monitored closely to ensure optimal recovery. Histological analyses On completion of behavioral observations, rats were overdosed with Nembutal and transcardially perfused with 4% paraformaldehyde (PFA). Extracted brains were post-fixed for 24 h in 4% PFA, followed by cryo-protection in a 30% sucrose solution in 0.1 M phosphate buffer solution and storage in 4 °C until sectioning. To verify cannula placements, brains were frozen and sliced into 50 μM coronal sections using a cryostat (LEICA CM1900). Coronal sections were mounted onto gel-coated slides and stained using cresyl violet. Cresyl violet staining Each slide was submerged in the following series of solutions for 2 min each: 100% ethanol, 95% ethanol, and 70% ethanol. Sections were then rinsed in distilled water to remove excess ethanol and subsequently placed in a 1% cresyl violet solution. Sections were then rinsed in distilled water to remove excess cresyl and immersed in 0.8% acetic acid to allow for differentiation (~2–5 min). Sections were then placed in 70%, 95% and 100% ethanol for 2 min each, respectively. Upon completion of staining, slides were immersed in Clearene solution for 15 min prior to cover-slipping with clarion mounting media. Microscopic examination was then used to determine location of infusion sites. Animals with incorrect cannulae placements were not included in the analyses. Experiment 1: the effects of chronic ghrelin on ad libitum food intake and body weight. The main goal of this experiment was to determine the effects of chronic intra-VTA ghrelin on food intake and body weight under standard ad libitum conditions. Twenty-four adult male rats (250–280 g) were single housed and received ad libitum chow and tap water. Following a one week baseline period measuring food intake and body weight, each rat received an indwelling unilateral guide cannula directed at the VTA. Each cannula was connected to an osmotic mini-pump delivering vehicle or one of two doses of ghrelin (3 nM, 10 nM). The rate of delivery was 0.5 μl/h/day for a total of 14 days. Food intake and body weight was monitored every 24 h throughout the infusion period, and for a week after the mini pumps were no longer delivering any drug. Experiment 2: the effects of chronic ghrelin on macronutrient diet preference. Given that ghrelin has been shown to preferentially increase the consumption of a high-fat diet (Shimbara et al., 2004), we hypothesized that ghrelin treated rats would choose high-fat as their primary source of calories, as well as show a substantial increase in body weight, compared to saline controls. Conversely, administration of a ghrelin antagonist was hypothesized to blunt the preference for the high-fat diet in turn causing a reduction in body weight gain.
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Twenty-six adult male rats (250–280 g) were single housed and exposed to three different diets simultaneously present in the home cage: 1) a carbohydrate rich diet, 2) a fat rich diet, and 3) a protein rich diet (Harlan–Teklad Diets; see Table 1 for information on caloric content). After baseline measurement of diet intake and body weight, animals were randomly assigned to 1 of 3 treatment groups and were implanted with 14-day osmotic mini-pumps delivering either 10 nM/ day of ghrelin (Peptides International; n = 9), 200 nM/day of a ghrelin antagonist ([D-Lys3]-GHRP-6; Peptides International; n = 9), or sterile saline (n = 8) into the VTA. Following surgery, daily intake of each macronutrient and body weight gain for the 14 days of treatment and 6 days post-infusion were recorded. Experiment 3: the effects of chronic ghrelin on progressive ratio responding for highly-palatable food pellets. To further examine the influence of ghrelin on the motivation to obtain a highly palatable food source, we measured how much work rats were willing to perform to obtain chocolate-flavored pellets that were isocaloric with our standard lab chow (Bioserv, Product # F0299; 3.68 kcal/g; see Table 2 for detailed content) using a progressive ratio (PR) operant paradigm. In a PR schedule experiment, the response contingency required to obtain a reward is progressively increased after each receipt of reinforcement. This paradigm requires that the animal increases the amount of effort to obtain the same magnitude of reward, thereby increasing the cost/benefit ratio. The efficacy of a given reinforcer (e.g. a food pellet) is defined in terms of the breakpoint (BP), the point in the series at which responding ceases (i.e. the final ratio completed by an animal) and reflects the maximum effort that the animal is willing to exert to obtain a particular reward (Hodos, 1961; Richardson and Roberts, 1996). Twenty-four adult male rats underwent caloric restriction and operant training procedures prior to surgery. To this end, animals were food restricted to 90% of their initial body weight and trained to bar press on a fixed-ratio 1 (FR1) schedule of responding in standard operant conditioning chambers (Colbourn Instruments) equipped with one nose-poke portal flanked by two retractable levers. Every correct press of the active lever resulted in the delivery of one 45 mg chocolate flavored food pellet. Each training session was 30 min in duration and was conducted between 0800 and 1200 h. Once each rat reached a stable level of responding on an FR1 schedule during training, defined as less than 15% variation in number of presses on 2 consecutive days during a 30 minute test session (~ 7 days), stereotaxic surgery was performed. During surgery, rats received minipumps filled with either saline (n = 8), ghrelin (10 nM/day; n = 8), or [D-Lys3]-GHRP-6 (n = 8). Rats were allowed to recover for seven days and tested on a PR schedule of responding on days 8, 10 and 14 following the surgery. The PR schedule required animals to increase the number of responses to obtain one food pellet after every correct response according to the following series: 1, 2, 4, 6, 9, 11, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219 etc. (from Richardson and Roberts, 1996). Table 1 Macronutrient composition of diets used in Experiment 2. Diet
Macronutrient
% by weight
% kcal from
Fat (60%)
Protein Carbohydrates Fat 5.1 Protein Carbohydrates Fat 4 Protein Carbohydrates Fat 3.7
23.5 27.3 34.3
18.4 21.3 60.3
17.7 70 5.2
17.8 70.4 11.8
60 21.1 5.2
64.7 22.7 12.6
Total kcal/g Carbohydrates (70%)
Total kcal/g Protein (60%)
Total kcal/g
Table 2 Macronutrient composition for operant pellets (45 mg). Purified chocolate pellets
Total kcal/g
Nutritional content
% by weight
Protein Carbohydrates Fat Fiber Ash Moisture
18.8 61.5 5.0 4.6 4.4 b5 3.68
We hypothesized that ghrelin treated rats would display an increased motivation to obtain chocolate-flavored pellets, as determined by an increase in breakpoints during treatment. Alternatively, we expected that [D-Lys3]-GHRP-6 treated rats would reduce their level of responding following treatment, suggesting an important role for ghrelin in the motivation to obtain palatable foods. Experiment 4: the effects of chronic intra-VTA ghrelin on operant responding in satiated rats. Here we examined the effects of ghrelin infusions into the VTA on operant responses in animals that were not hungry when tested. Twenty adult male rats (250–280 g) were singlehoused and received ad libitum chow and water during the acclimatization week, during which baseline food intake and body weight were recorded. Rats were subsequently calorically restricted to ~ 50% of their daily food intake until they reached 90% of their body weight. Then, rats received daily 30 minute training sessions until they reached a stable level of responding on a FR1 schedule of reinforcement. Animals were then implanted with intra VTA cannulae attached to mini-pumps filled with either saline (n = 10) or ghrelin (n = 10, 10 nM/day). Following surgical procedures, animals were returned to ad libitum feeding and food intake and body weight were recorded daily. On day 4 post-surgery rats were tested in the operant chambers on an FR 1 schedule to allow comparison to baseline levels of responding, when animals were food restricted. Results Animals in all studies showed minimal discomfort as a result of the surgical procedures and none of the animals had to be discarded due to illness or surgical or post surgical complications. A representative cannulae placement photo and animals that were excluded due to incorrect placements are outlined in Fig. 1. Experiment 1. Ghrelin infused chronically into the VTA increases food intake and body weight gain A one-way between groups ANOVA revealed that by the end of the baseline period, all rats were consuming a similar amount of chow per day (F(2,18) = 1.452, p N 0.05; data not shown) and displayed no significant differences in body weight (F(2,18) = .586, p N 0.05; data not shown). Given these data we organized and analyzed experimental data in the form of change from baseline scores. Following surgery we observed a dose response effect of ghrelin delivered chronically into the VTA on food intake (F(2,18) = 4.337, p b 0.05). Rats receiving the 10 nM dose of ghrelin consumed an average of 6.2 g more chow per day during treatment compared to baseline consumption, whereas saline treated rats consumed only 2.92 g more chow per day (p b 0.05; see Fig. 2a). The group infused with 3 nM ghrelin ate an average of 4.27 g more chow per day, but this change from baseline did not differ significantly from that observed in either high ghrelin (p = .090) or saline groups (p N 0.05; see Fig. 2a). Similarly, chronic intra VTA ghrelin delivery resulted in a significant increase in weight gain from the baseline that was dose dependent (see Fig. 2b). When total weight gain over the infusion period was examined using a one-way ANOVA, rats treated with the
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Fig. 1. Representative photomicrograph of cannula placements in the ventral tegmental area (VTA) and table outlining number of animals discarded from analysis in each experiment based on incorrect placements.
high dose of ghrelin exhibited the greatest total weight gain from baseline (F(2,18) = 3.743, p b 0.05; M = 99.33 g), significantly different from the saline group (p = .021; M = 81.71 g), but not different from the low ghrelin group (p b 0.05; M = 86.75 g; see Fig. 2b).
the preferred high fat diet (F(2,18) = 172.780, p b 0.05), relative to both saline (p b .05) and ghrelin treated rats (p b 0.05 Tukey's HSD, see Fig. 3c). Neither ghrelin nor [D-Lys3]-GHRP-6 infusions affected the intake of the protein or carbohydrate rich diet (p N 0.05; See Fig. 3c).
Experiment 2. GHSR blockade in the VTA decreases the intake of food with a high caloric content coming from fat
Experiment 3. Ghrelin infusions into the VTA enhance, while GHSR blockade prevents increases in food motivation as measured by progressive ratio responding under restricted feeding conditions
As in the previous study, there were no differences in food intake or body weight between the groups during the baseline period (p N 0.05; data not shown). It should be noted that all animals showed a clear preference for the diet with high caloric content coming from fat (Fig. 3a). In addition, access to a choice in diets resulted in high caloric intake, and a rapid gain in weight in all animals (Raynor and Epstein, 2001), in comparison with similar baseline measures from animals in the first experiment (See Table 3). Interestingly, while intra-VTA ghrelin did not further increase neither caloric intake nor body weight gain in this experiment, the blockade of ghrelin receptors in the VTA with the ghrelin antagonist [D-Lys3]-GHRP-6 selectively attenuated their body weight gain (F(2,18) = 9.32, p b 0.05 Tukey's HSD; see Fig. 3b) and their intake of
A one way ANOVA showed that rats in each group had similar body weights before any experimental manipulations were conducted (F(2,19) = .812, p N 0.05; data not shown). Similarly, there were no differences between rats in the time it took to acquire a stable rate of operant responding during the training period (p N 0.05). Following surgery, animals were tested under the progressive ratio schedule of responding at three different time points during the infusion period: 1) On day 8 of the infusion period (day 1 of food restriction); 2) day 11 of the infusion period (day 4 food restriction); and 3) day 14 of the infusion period (day 7 of food restriction). All animals were tested under restricted feeding conditions that began on the evening of day 7 postinfusion. As shown by repeated measures ANOVA (time interval x group), by day 14 there was an overall treatment effect where ghrelin treated rats responded more than saline or antagonist treated rats (main effect for treatment, F(1,19) = 3.741, p b .05). As expected, efforts to obtain pellets were increased over time following the food restriction regimen (main effect for time interval, F(1,19) = 22.798, p b 0.05). a significant interaction effect emerged, demonstrating that rats infused with the ghrelin receptor antagonist into the VTA, failed to increase their efforts to obtain pellets over time in spite of being food restricted, in comparison to ghrelin treated animals (F(2,19) = 6.599, p b 0.05, followed by post hoc Tukey's HSD: p b 0.05; see Fig. 4). The data were also analyzed in terms of break points, defined as the last completed (reinforced) ratio before the animal stopped responding. As shown in Fig. 5, a repeated measures ANOVA revealed no differences between the groups on day 1 or day 4 following the onset of food restriction (p N 0.05; data not shown). However, during the PR session on day 7, differences were evident between groups (F(2,19) = 7.398, p b .05), with the ghrelin infused rats showing an increase in effort spent to obtain chocolate-flavored pellets relative to the [D-Lys3]-GHRP-6 group (p b 0.05). Ghrelin treated rats pressed the reinforcing lever 167 times on average to receive one chocolate pellet, whereas [D-Lys3]-GHRP-6 treated rats pressed the lever only 42.5 times on average to receive a similar reward. Experiment 4. Ghrelin infusions into the VTA produce “hungry-like” rates of reinforcement in satiated animals
Fig. 2. Mean (+/−SEM) change from baseline on a) daily food intake and b) body weight gain per day in animals infused with either saline (n = 7), 3 nM ghrelin (n = 8), or 10 nM ghrelin (n = 6) during the infusion period (n = 6). * p b .05.
Animals trained to bar press for food under restricted feeding conditions were implanted with cannulae delivering saline or ghrelin
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Fig. 3. Panel a depicts the Mean (+/−SEM) daily kilocalorie consumption as a function of macronutrient diet during the baseline period. All animals exhibited a clear preference for the diet with high caloric content coming from fat over the other diets. Panel b depicts weight gain during the infusion period, clearly showing an attenuation in weight gain in animals infused with [D-Lys3]-GHRP-6. * p b .05. Panel c shows the mean (+/−SEM) daily caloric intake of the high fat, high protein, high carbohydrate diet, and the total caloric intake during the infusion period. ([D-Lys3]-GHRP-6: n = 8; saline: n = 6; ghrelin: n = 8). As seen in this panel, GHSR antagonism with [D-Lys3]-GHRP-6 attenuated high fat intake throughout the infusion period relative to control and ghrelin treated rats. No differences were found between groups on measures of high-protein or high-carbohydrate diets during treatment (p b 0.05).
(10 nM) into the VTA. By the end of infusion period, ghrelin treated rats had consumed more chow during the treatment period compared to saline treated rats (t(1,15) = −3.170, p b .05; Fig. 5a), consistent with greater total body weight gain over the two week period (t(1,15) = −2.195, p b .05; Fig. 6) (see Fig. 6b). When tested in the operant boxes, ghrelin treated rats with full access to food at all times after surgery showed a rate of responding that Table 3 Comparison of daily caloric intake and bodyweight during baseline.
Experiment 1 (standard chow) Experiment 2 (3 diets)
Daily chow intake (kcal)
Weight gain
98.45 +/− 1.789 135.89 +/− 3.65
70.38 +/− 1.81 41.63 +/− 2.72
was nearly equal (about 86%) to that of their baseline rates during which they were food restricted. In contrast, saline treated rats responded at a much lower rate (about 34%) of their baseline “hungry” rate on an FR1 schedule. The difference between ghrelin and vehicle treated rats was statistically significant (t(1,15) = −2.558, p b .05; see Fig. 6c). Discussion In the present series of studies we examined the VTA as a target for ghrelin to enhance the intake of and motivation to obtain palatable food. Using mini-osmotic pumps we chronically infused ghrelin into the VTA of rats, and this treatment resulted in a dose dependant increase in food intake and body weight compared to vehicle infused rats. Furthermore, chronic infusions of a ghrelin antagonist into the
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Fig. 4. Mean (+/−SEM) cumulative bar presses throughout each of the progressive ratio testing sessions during the infusion period. ([D-Lys3]-GHRP-6: n = 7; saline: n = 8; ghrelin: n = 7). By the last day of testing, ghrelin treated animals were pressing the active lever more frequently than both the antagonist and saline treated rats. Antagonist treated rats failed to show an increase in responding across the infusion period compared to ghrelin treated rats, despite both groups being food restricted.
VTA selectively decreased the intake of a preferred high-fat diet, and attenuated the extreme body weight gain that is seen in control animals that had access to a variety of diets. Chronic VTA infusions of the antagonist also decreased motivation to work for chocolate flavored (but not calorie rich) pellets in hungry animals, whereas ghrelin further enhanced motivation to work for these pellets in hungry animals. Finally, animals receiving ghrelin infusions into the VTA responded like hungry animals when tested on an FR1 schedule under ad lib conditions.
Fig. 5. Amount of work rats were willing to perform as measured by Mean (+/−SEM) breakpoints as observed in each progressive ratio testing session. During the final progressive ratio test ghrelin treated rats expended greater effort to obtain chocolate pellets relative to the antagonist treated rats. * p b .05.
Previous studies have shown that chronic ghrelin infusions, both peripheral and central (intracerebroventricular), result in increased food intake, body weight and adiposity in rodents (Perez-Tilve et al., 2011; Tschop et al., 2000). While these effects have been generally attributed to the actions of ghrelin on receptors within the hypothalamic arcuate nucleus, our current data suggest that chronic VTA stimulation with ghrelin can produce similar results. In fact, the body weight gain and food intake seen in our study following chronic VTA infusions of ghrelin, are very similar to those reported by Tschop et al. (2000) in rats chronically infused with ghrelin into the ventricles. These data further support a number of recent studies demonstrating that acute ghrelin infusions directed as the VTA results in an increased motivation for palatable food in mice as measured by CPP and operant paradigms (Egecioglu et al., 2010; Landgren et al., 2011; Skibicka et al., 2011, in press). While these data do not imply that the VTA is uniquely responsible for the obesogenic effects of ghrelin, they do indicate that the VTA could play an important role in the regulation of food intake and body weight. These data also confirm that ghrelin stimulates appetite by acting directly on the VTA (Abizaid et al., 2006; Naleid et al., 2005). Furthermore, these data suggest that these effects can persist in spite of chronic stimulation of GHSRs. This was clearly evident in rats given the highest dose of ghrelin, who were eating about 30% (an average of 9 g of food) more on the last day of ghrelin infusion than their baseline average intake. Control animals also increased their intake of food (something that is natural in animals that are growing), but this increase was less substantial at 12% (4.5 g on average). Thus, even in the absence of caloric restriction, chronically elevated ghrelin can serve to induce a sustained feeding response when administered into the VTA.
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Fig. 6. Mean (+/−SEM) a) body weight gain; b) cumulative chow consumption, and c) percent of baseline responding following surgery on an FR1 schedule of reinforcement during the infusion period (Saline: n = 7; Ghrelin: n = 10). Ghrelin treated rats gained more weight, ate more, and bar-pressed for food more under ad lib conditions than saline infused animals. * p b .05.
Our study supports the notion that ghrelin receptors in the VTA mediate the consumption of preferred foods, whether the preference is for calorie rich high fat diet or an isocaloric chocolate flavored food pellet. We found, as others have previously (see Raynor and Epstein, 2001 for review), that our rats consumed a large amount of calories (approximately 70% more) and gained more weight when given access to a variety of foods compared to rats given regular chow. The rats in our study showed a strong preference for the food with the highest percentage of calories coming from fat. While ghrelin infusions into the VTA were not effective in further increasing neither total caloric intake, nor the intake of the high fat selectively, infusions of the ghrelin antagonist selectively reduced the intake of this high fat diet without altering the intake of the other two diets. Similarly, while intra VTA ghrelin did not further affect weight gain, the antagonist attenuated weight gain during the infusion period. It should be noted, however, that the antagonist utilized in this series of studies has been shown to be less specific to the GHSR than some alternatives (Depoortere et al., 2006). Nevertheless, our results are consistent
with those of others using different antagonists to reduce behavioral responses to obtain food and other reinforcers (Egecioglu et al., 2010; Landgren et al., 2011; Skibicka et al., 2011, in press). Previous human and animal studies have shown that ghrelin not only increases food intake but also that, when given a choice, ghrelin treatment increases the intake of preferred foods via central mechanisms. For example, ICV ghrelin infusions increased the intake of a highfat diet that was presented to rats simultaneously with a highcarbohydrate diet (Shimbara et al., 2004). In rats and mice, ghrelin injections increase the intake of sweet tasting solutions regardless of caloric content, an effect not seen in ghrelin insensitive mice (Disse et al., 2010). In contrast, ghrelin receptor antagonists decrease the intake of palatable liquid diets in rats (e.g. Ensure™), while acute intra VTA infusions increase the intake of these same diets (Egecioglu et al., 2010). Human studies have shown that hunger scores and food intake increase after ghrelin injections (Wren et al., 2001b), and that ghrelin can selectively increase imagery of preferred foods regardless of the type of food (Schmid et al., 2005). Moreover, ghrelin stimulates the mesolimbic reward system of human subjects that are shown images of food (Malik et al., 2008). It is likely that ghrelin acts on the VTA to increase the incentive value of food, particularly preferred foods if these are present. Further, our data suggest that the VTA may be a potential pharmaceutical target for ghrelin related drugs to reduce overconsumption of palatable foods, or increase food intake in clinical conditions where this is required. Given that systemically and centrally administered ghrelin acts on the VTA to stimulate DA cells and increases extracellular concentration of DA in the NAc (Abizaid et al., 2006; Jerlhag et al., 2007; Quarta et al., 2009), we hypothesized that ghrelin administration into the VTA would increase instrumental responses for a reward. Our results confirmed this. In Experiment 3 we show that vehicle treated animals generally increased the number of responses they were willing to perform as the duration of food restriction increased. Ghrelin infused rats showed similar responses as vehicle treated rats after one and four days of food restriction, but were more willing to continue responding in the face of increased demands seven days after the food restriction began. In contrast, animals chronically infused with the ghrelin antagonist never increased their willingness to respond to the increased demands posed by the progressive ratio schedule. Interestingly, even after seven days of food restriction, antagonist treated rats quit responding sooner than vehicle treated rats that, in turn, had a shorter latency to quit responding than ghrelin treated rats. While we did not observe a ghrelin effect on PR responses when rats were not food restricted, we did observe that ghrelin infused into the VTA made satiated rats respond at the same rate or higher than they did when they were hungry under an F1 schedule. In contrast, vehicle treated rats responded only at about 60% of their hungry rates, indicating that ghrelin may act on the VTA to produce some behavioral responses in satiated animals that are akin to those seen in hungry animals. The role of the VTA in the modulation of feeding is complex, and appears to be closely linked to the motivational aspects of feeding, in addition to influencing the hunger/satiety pathway that is generally governed by the hypothalamus. The relative contribution of the hypothalamus and the VTA in the central mechanisms that govern feeding behavior and motivation remains to be fully determined. Nevertheless, it is possible that ghrelin receptors in the VTA enhance information that is relayed to the VTA via a number of ascending and descending pathways that have been implicated in arousal, sensory information, and reward. For instance, ghrelin receptors in the VTA may facilitate the activation of DA neurons by orexin, a lateral hypothalamic peptide that generates arousal and has been linked to reward seeking behaviors (Aston-Jones et al., 2010; Boutrel et al., 2010; Thompson and Borgland, 2010). Similarly, ghrelin receptors in the VTA may lower the threshold of activation mediated by glutamatergic and/or cholinergic inputs to the VTA and influence the intake of rewarding food (Abizaid et al., 2006; Jerlhag et al., 2006).
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Moreover, recent reports point to the importance of the interactions between cholinergic pathways and ghrelin signaling pathways to influence the VTA (Dickson et al., 2010; Disse et al., 2011). The experimental approach used in the present study allowed us to deliver ghrelin or ghrelin antagonists directly onto the VTA of rats at a constant rate for a period of 14 days. Postmortem histological examination determined that most of the animals in each study had cannulae that were accurate in their placement. Indeed, very few animals per experiment had a cannula that was in areas surrounding the VTA, and these animals were discarded from further analyses. We cannot discard the possibility that the observed effects of the different drugs infused with the minipumps are due at least in part to the actions of these drugs spilling into nearby areas. Previous studies, however, have shown that acute infusions of ghrelin at a rate of 0.5 μl infused over a period of two minutes into the rat VTA do not produce spillage outside of the VTA (Abizaid et al., 2006). Furthermore, in these same acute feeding experiments, ghrelin infusions into areas surrounding the VTA failed to produce any significant effects on feeding (Abizaid et al., 2006). Thus, given these data we attribute our results to the effects of ghrelin or its antagonists to their actions on GHSR within the VTA. As chronic overeating and the resultant obesity that often accompanies it has become an increasing threat to the health of individuals worldwide, it is important to determine what can be done to reduce it. As we have shown, antagonism of ghrelin not only reduced body weight gain and preference for a high-fat diet when no effort was required, it also blunted the effect of ghrelin on the effort expended to obtain flavored food pellets. Emerging evidence, including the current study, suggests that the ghrelin system may provide a potential therapeutic target for the treatment of obesity or wasting disorders. Acknowledgments This project was funded by a Natural Science and Engineering Research Council of Canada (NSERC) Discovery grant (AA), an Ontario Graduate Scholarship (SJK), and NSERC summer undergraduate scholarships (EO and AI). References Abizaid, A., Liu, Z., Andrews, Z.B., Shanabrough, M., Borok, E., Elsworth, J.D., Roth, R.H., Sleeman, M.W., Picciotto, M.R., Tschop, M., Gao, X., Horvath, T., 2006. Ghrelin modulates the activity and synaptic input organization of midbrain dopamine neurons while promoting appetite. J. Clin. Invest. 116 (12), 3229–3239. Aston-Jones, G., Smith, R.J., Sartor, G.C., Moorman, D.E., Massi, L., Tahsili-Fahadan, P., Richardson, K.A., 2010. Lateral hypothalamic orexin/hypocretin neurons: a role in reward-seeking and addiction. Brain Res. 1314, 74–90. Blum, I.D., Patterson, Z., Khazall, R., Lamont, E.W., Sleeman, M.W., Horvath, T.L., Abizaid, A., 2009. Reduced anticipatory locomotor responses to scheduled meals in ghrelin receptor deficient mice. Neuroscience 164 (2), 351–359. Boutrel, B., Cannella, N., de Lecea, L., 2010. The role of hypocretin in driving arousal and goal directed behaviors. Brain Res. 1314, 103–111. Clifford, S., Zeckler, R.A., Buckman, S., Thompson, J., Wellman, P.J., Smith, R.G., 2011. Impact of food restriction and cocaine on locomotion in ghrelin- and ghrelinreceptor knockout mice. Addict. Biol. 16 (3), 386–392 (Jul). Cummings, D.E., Purnell, J.Q., Frayo, R.S., Schmidova, K., Wisse, B.E., Weigle, D.S., 2001. A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 50, 1714–1719. Cummings, D.E., Frayo, R.S., Marmonier, C., Aubert, R., Chapelot, D., 2004. Plasma ghrelin levels and hunger scores in humans initiating meals voluntarily without time- and food-related cues. Am. J. Physiol. Endocrinol. Metab. 287, 297–304. Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., Suganuma, T., Matsukura, S., Kangawa, K., Nakazato, M., 2000. Ghrelin, a novel growth hormone releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141 (11), 4255–4261. Date, Y., Shimbara, T., Koda, S., Toshinai, K., Ida, T., Murakami, N., Miyazato, M., Kokame, K., Ishizuka, Y., Ishida, Y., Kageyama, H., Shioda, S., Kangawa, K., Nakazoto, M., 2006. Peripheral ghrelin transmits orexigenic signals through the noradrenergic pathway from the hindbrain to the hypothalamus. Cell Metab. 4, 323–331. Depoortere, I., Thijs, T., Peeters, T., 2006. The contractile effect of the ghrelin receptor antagonist, D-Lys-3 [GHRP-6], in rat fundic strips is mediated through 5-HT receptors. Eur. J. Pharmacol. 537, 160–165. Dickson, S.L., Hrabovsky, E., Hansson, C., Jerlhag, E., Alvarez-Crepo, M., Skibicka, K.P., Molnar, C.S., LIposits, Z., Engel, J., Egecioglu, E., 2010. Blockade of central nicotine
579
acetylcholine receptor signaling attenuates ghrelin-induced food intake in rodents. Neuroscience 171, 1180–1186. Disse, E., Bussier, A.L., Veyrat-Durebex, C., Deblon, N., Pfluger, P.T., Tschop, M.H., Laville, M., Rohner-Jeanrenaud, F., 2010. Peripheral ghrelin enhances sweet taste food consumption and preference, regardless of its caloric content. Physiol. Behav. 101 (2), 277–281. Disse, E., Bussier, A.L., Deblon, N., Pfluger, P.T., Tschop, M.H., Laville, M., RohnerJeanrenaud, F., 2011. Systemic ghrelin and reward: effect of cholinergic blockade. Physiol. Behav. 102, 481–484. Drazen, D.L., Vahl, T.P., D'Alessio, D.A., Seeley, R.J., Woods, S.C., 2006. Effects of a fixed meal pattern on ghrelin secretion: evidence for a learned response independent of nutrient status. Endocrinology 147 (1), 23–30. Egecioglu, E., Jerlhag, E., Salome, N., Skibicka, K.P., Haage, D., Bohlooly-Y, M., Andersson, D., Bjursell, M., Perrissoud, D., Engel, J., Dickson, S.L., 2010. Ghrelin increases intake of rewarding food in rodents. Addict. Biol. 15 (3), 304–311. Faulconbridge, L.F., Cummings, D.E., Kaplan, J.M., Grill, H.J., 2003. Hyperphagic effects of brainstem ghrelin administration. Diabetes 52, 2260–2265. Guan, X.M., Yu, H., Palyha, O.C., McKee, K.K., Feighner, S.D., Sirinathsinghji, D.J., Smith, R. G., Van der Ploeg, L.H.T., Howard, A.D., 1997. Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Mol. Brain Res. 48, 23–29. Hodos, W., 1961. Progressive Ratio as a measure of reward strength. Science 134, 943–944. Jerlhag, E., Egecioglu, E., Landgren, S., Salome, N., Heilig, M., Moechars, D., Datta, R., Perrissoud, D., Dickson, S.L., Engel, J.A., 2006. Ghrelin stimulates locomotor activity and accumbal dopamine-overflow via central cholinergic systems in mice: implications for its involvement in brain reward. Addict. Biol. 11 (1), 45–54 (Mar). Jerlhag, E., Egecioglu, E., Dickson, S.L., Douhan, A., Svensson, L., Engel, J.A., 2007. Ghrelin administration into tegmental areas stimulates locomotor activity and increases extracellular concentration of dopamine in the nucleus accumbens. Addict. Biol. 12, 6–16. Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., Kangawa, K., 1999. Ghrelin is a growth-hormone releasing acylated peptide from stomach. Nature 402, 656–660. Landgren, S., Simms, J.A., Thelle, D.S., Strandhagen, E., Bartlett, S.E., Engel, J.A., Jerlhag, E., 2011. The ghrelin signalling system is involved in the consumption of sweets. PLoS One 6 (3), e18170. LeSauter, J., Hoque, N., Weintraub, N., Pffaf, D.W., Silver, R., 2009. Stomach ghrelinsecreting cells as food-entrainable circadian clocks. PNAS 106 (32), 13582–13587. Malik, S., McGlone, F., Bedrossian, D., Dagher, A., 2008. Ghrelin modulates brain activity in areas that control appetitive behavior. Cell Metab. 7 (5), 400–409. Nagaya, N., Uematsu, M., Kojima, M., Date, Y., Nakazato, M., Okumura, H., Hosoda, H., Shimizu, W., Yamagishi, M., Oya, H., Yutani, C., Kangawa, K., 2001. Elevating circulating levels of ghrelin in cachexia associated with chronic heart failure: relationships between ghrelin and anabolic/catabolic factors. Circulation 104, 2034–2038. Nakazato, M., Murakami, N., Date, Y., Kojima, M., Matsuo, H., Kangawa, K., Matsukura, S., 2001. A role for ghrelin in the central regulation of feeding. Nature 409, 194–198. Naleid, A.M., Grace, M.K., Cummings, D.E., Levine, A.S., 2005. Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens. Peptides 26, 2274–2279. Paxinos, G., Watson, C., 1998. The rat brain in stereotaxic coordinates, 4th edition. Academic Press, San Diego, CA. Perello, M., Sakata, I., Birnbaum, S., Chuang, J.C., Osbourne-Lawrence, S., Rovinsky, S.A., Woloszyn, J., Yanagisawa, M., Lutter, M., Zigman, J., 2009. Ghrelin increases the rewarding value of high-fat diet in an orexin dependent fashion. Biol. Psychiatry 67 (9), 880–886. Perez-Tilve, D., Heppner, K., Kirchner, F., Lockie, S.H., Woods, S.C., Smiley, D.L., Tschop, M., Pfluger, P., 2011. Ghrelin induced adiposity is independent of orexigenic effects. FASEB J. 25 (8), 2814–2822 (Aug). Quarta, D., DiFrancesco, C., Melotto, S., Mangiarini, L., Heidbreder, C., Hedou, G., 2009. Systemic administration of ghrelin increases extracellular dopamine in the shell but not the core subdivision of the nucleus accumbens. Neurochem. Int. 54 (2), 89–94. Raynor, H.A., Epstein, L.H., 2001. Dietary variety, energy regulation and obesity. Psychol. Bull. 127 (3), 325–341. Richardson, N.R., Roberts, D.C.S., 1996. Progressive ratio schedules in drug selfadministration studies in rats: a method to evaluate reinforcing efficacy. J. Neurosci. Methods 66, 1–11. Schmid, D.A., Held, K., Ising, M., Uhr, M., Weickel, J.C., Steiger, A., 2005. Ghrelin stimulates appetite, imagination of food, GH, ACTH, and cortisol, but does not affect leptin in normal controls. Neuropsychopharmacology 30, 1187–1192. Shimbara, T., Mondal, M.S., Kawagoe, T., Toshinai, K., Koda, S., Yamaguchi, H., Date, Y., Nakazato, M., 2004. Central administration of ghrelin preferentially increases fat ingestion. Neurosci. Lett. 369, 75–79. Skibicka, K.P., Hansson, C., Alvarez-Crespo, M., Friberg, P.A., Dickson, S.L., 2011. Ghrelin directly targets the ventral tegmental area to increase food motivation. Neuroscience 180, 129–137 (Apr 28). Skibicka, K.P., Hansson, C., Egecioglu, E., Dickson, S.L., in press. Role of ghrelin in food reward: impact of ghrelin on sucrose self-administration and mesolimbic dopamine and acetycholine receptor gene expression. Addict. Biol. doi:10.1111/j. 1369-1600.2010.00294.x. Thompson, J.L., Borgland, S.L., 2010. A role for orexin/hypocretin in motivation. Behav. Brain Res. 217, 446–453. Toshinai, K., Mondal, M.S., Nakazato, M., Date, Y., Murakami, N., Kojima, M., Kangawa, K., Matsukura, S., 2001. Upregulation of ghrelin expression in the stomach upon
580
S.J. King et al. / Hormones and Behavior 60 (2011) 572–580
fasting, insulin induced hypoglycemia and leptin administration. Biochem. Biophys. Res. Commun. 281 (5), 1220–1225. Tschop, M., Smiley, D.L., Heiman, M.L., 2000. Ghrelin induces adiposity in rodents. Nature 407, 908–913. Tschop, M., Wawarta, R., Reipel, R.L., Freidrich, S., Bindlingmaier, M., Landgraf, R., Folwaczny, C., 2001. Postprandial decrease of circulating ghrelin human ghrelin levels. J. Endocrinol. Invest. 24 (6), 19–21. Weinberg, Z.Y., Nicholson, M.L., Currie, P.J., 2011. 6-hydroxydopamine lesions of the ventral tegmental area suppress ghrelin's ability to elicit food reinforced behavior. Neurosci. Lett. 499, 70–73. Willesen, M.G., Kristensen, P., Romer, J., 1999. Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Neuroendocrinology 70 (5), 306–316.
Wren, A.M., Small, C.J., Ward, H.L., Murphy, K.G., Dakin, C.L., Taheri, S., Kennedy, A.R., Roberts, G.H., Morgan, D.G., Ghatei, M.A., Bloom, S.R., 2000. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Reg. Pept. 140, 148–152. Wren, A.M., Seal, L.J., Cohen, M.A., Brynes, A.E., Frost, G.S., Murphy, K.G., Dhillo, W.S., Ghatei, M.A., Bloom, S.R., 2001a. Ghrelin enhances appetite and increases food intake in humans. J. Clin. Endocrinol. Metab. 86 (12), 5992–5995. Wren, A.M., Small, C.J., Abbott, C.R., Dhillo, W.S., Seal, L.J., Cohen, M.A., Batterham, R.L., Taheri, S., Stanley, S.A., Ghatei, M.A., Bloom, S.R., 2001b. Ghrelin causes hyperphagia and obesity in rats. Diabetes 50, 2540–2547. Zigman, J.M., Jones, J.E., Lee, C.E., Saper, C.B., Elmquist, J.K., 2006. Expression of the ghrelin receptor mRNA in the rat and the mouse brain. J. Comp. Neurol. 494, 528–548.